The biopharmaceutical industry strives for improvement of their production processes. In recent years, miRNAs have been shown to positively impact the production capacity of recombinant CHO cells, especially with regard to difficult to express proteins. Effective and reliable gene regulation of process relevant target genes by miRNAs is a prerequisite for integrating them into the toolbox of industrial cell engineering strategies. However, most studies rely on transient transfection of miRNA mimics; there is low standardization in evaluation of miRNA function and little knowledge on transferability of effects found during transient expression to stable expression during industry relevant fed‐batch cultivation. In order to provide more insight into this topic, we used the pcDNA6.2 vector for stable miRNA overexpression during batch and fed‐batch cultivation in CHO DG44 cells, optimized the vector, and compared the miRNA levels and effects with those achieved by transfection of miRNA mimics. We found that miR‐1 downregulated TWF1 mRNA in different recombinant CHO DG44 clones in a dose‐dependent manner during transient batch cultivation. Cells stably overexpressing miR‐1 also showed a TWF1 mRNA downregulation when cultivated in batch mode using in‐house medium 1. However, when the cells stably overexpressing miR‐1 were cultivated in fed‐batch mode using in‐house medium 2. Consequently, a change of cultivation mode and medium seems to have an impact on target gene regulation by miRNA. Taken together, our findings highlight the importance to standardize miRNA evaluations and test miRNAs in the final application environment.

中文翻译：

---

miRNA 技术在生物加工中的可转移性：培养模式和培养基的影响

生物制药行业致力于改进其生产工艺。近年来，已显示 miRNA 对重组 CHO 细胞的生产能力产生积极影响，特别是在难以表达的蛋白质方面。miRNA对过程相关靶基因的有效和可靠的基因调控是将它们整合到工业细胞工程策略工具箱中的先决条件。然而，大多数研究依赖于 miRNA 模拟物的瞬时转染。miRNA 功能评估的标准化程度低，对于工业相关流加培养过程中瞬时表达期间发现的效应到稳定表达的可转移性知之甚少。为了更深入地了解这个主题，我们使用了 pcDNA6。2 用于在 CHO DG44 细胞分批和补料分批培养期间稳定 miRNA 过表达的载体，优化了载体，并将 miRNA 水平和效果与转染 miRNA 模拟物所获得的水平和效果进行了比较。我们发现，在瞬时分批培养过程中，miR-1 在不同的重组 CHO DG44 克隆中以剂量依赖性方式下调 TWF1 mRNA。当使用内部培养基 1 以分批模式培养时，稳定过表达 miR-1 的细胞也显示出 TWF1 mRNA 下调。然而，当使用内部培养基 2 以分批补料模式培养稳定过表达 miR-1 的细胞时。因此，培养模式和培养基的改变似乎对miRNA对靶基因的调控产生了影响。总之，我们的研究结果强调了在最终应用环境中标准化 miRNA 评估和测试 miRNA 的重要性。